L-695,256 is a synthetic carbapenem beta-lactam antibiotic that binds with a high degree of affinity to penicillin-binding protein (PBP) PBP 2a, the protein that mediates staphylococcal resistance to methicillin. The concentration of L-695,256 that inhibited binding of radiolabeled [3H]penicillin to PBP 2a by 50% was 1.2 micrograms/ml, whereas they were 14 and 68 micrograms/ml for penicillin and imipenem, respectively. Cell wall synthesis, determined by incorporation of [14C]N-acetylglucosamine into whole cells, was inhibited by 50% at concentrations of 1.3, 26, and 132 micrograms/ml for L-695,256, penicillin, and imipenem, respectively, for the methicillin-resistant strain COL. Growth of cells of each of two homogeneously resistant strains, COL and 76, was completely inhibited by 4 micrograms of L-695,256 per ml, whereas growth was inhibited by 100 micrograms or more of penicillin or imipenem per ml. The efficacies of L-695,256 (10 mg/kg given three times daily [t.i.d.]), imipenem (37.5 mg/kg t.i.d.), penicillin (300,000 units/kg t.i.d.), and vancomycin (25 mg/kg given twice daily) were compared in the rabbit model of aortic valve endocarditis established with these homogeneous strains. After 4 days of treatment, mean bacterial densities in aortic valve vegetations were reduced by 4.0 to 5.8 log10 CFU/g for L-695,256, 1.0 to 1.8 log10 CFU/g for imipenem, -1.1 to 3.9 log10 CFU/g for penicillin, and 1.1 to 3.0 log10 CFU/g for vancomycin in comparison to the densities of controls. Compounds such as L-695,256 that are bound by PBP 2a with a high degree of affinity are likely to be extremely effective in the treatment of infections caused by methicillin-resistant staphylococci.